scholarly journals Validation of Reference Genes for Gene Expression Normalization in RAW264.7 Cells under Different Conditions

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Zhenzhen Bao ◽  
Yanli Huang ◽  
Jiyu Chen ◽  
Zhenglong Wang ◽  
Jiang Qian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Raw264.7 Cells ◽  
Antigen Uptake ◽  
Expression Levels ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

RAW264.7 is a macrophage strain derived from mice tumour and shows a significant ability in antigen uptake. Real-time quantitative PCR (RT-qPCR) is one of the most commonly used methods in gene studies and requires suitable reference genes to normalize and quantitate the expression of gene of interest with sensitivity and specificity. However, suitable reference genes in RAW264.7 cells have not yet been identified for accurate gene expression quantification. In the current study, we evaluated expression levels of ten candidate reference genes in RAW264.7 cells under different conditions. RT-qPCR results indicated significant differences in the expression levels among the ten reference genes. Statistical analyses were carried out using geNorm, NormFinder, and BestKeeper software to further investigate the stability of the reference genes. Integrating the results from the three analytical methods, cytochrome c-1 and hydroxymethylbilane synthase were found to be the most stable and therefore more suitable reference genes, while ribosomal protein L4 and cyclophilin A were the least stable. This study emphasises the importance of identifying and selecting the most stable reference genes for normalization and provides a basis for future gene expression studies using RAW264.7 cells.

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

scholarly journals Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorota M. Krzyżanowska ◽  
Anna Supernat ◽  
Tomasz Maciąg ◽  
Marta Matuszewska ◽  
Sylwia Jafra
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Reference Genes ◽  
Significance Threshold ◽  
Expression Studies ◽  
Reverse Transcription Quantitative Pcr ◽  
Gene Expression Studies ◽  
The Stability ◽  
Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

scholarly journals Identification of Suitable Reference Genes for gene Expression Studies by qRT-PCR in the Blister BeetleMylabris cichorii

2014 ◽  
Vol 14 (94) ◽  
pp. 1-10 ◽  
Author(s):  
Yu Wang ◽  
Zhong-Kang Wang ◽  
Yi Huang ◽  
Yu-Feng Liao ◽  
You-Ping Yin
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference

scholarly journals In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

2007 ◽  
Vol 8 (1) ◽  
pp. 47 ◽  
Author(s):  
Monika Jung ◽  
Azizbek Ramankulov ◽  
Jan Roigas ◽  
Manfred Johannsen ◽  
Martin Ringsdorf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Real Time Pcr ◽  
Renal Cell ◽  
Reference Genes ◽  
Human Renal Cell Carcinoma ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference

Determination of Suitable Candidate Genes as Endogenous Control for Gene Expression Analysis in Local Capsicum Annuum MC11

2021 ◽  
Vol 12 (3) ◽  
pp. 1011-1017
Author(s):  
Marina Mokhtar Et.al
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Capsicum Annuum ◽  
Reference Genes ◽  
Data Normalization ◽  
Plant Parts ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for qRT-PCR assays. This study identifies suitable reference genes for local chilli, Capsicum annuum var MC11 under incident of Cucumber mosaic virus infection. Six candidate genes actin, tub, EF1α, GAPDH, TEF1α and 18SrRNA and three validated Capsicum reference genes UBI-3 ref, β-tub ref and gapdhref were tested against five chilli plant parts stem, shoot, leave, flower and root.  The PCR/qRT-PCR results demonstrate only five candidate references genes actin, EF1α, GAPDH, 18SrRNA, and TEF1α that show specific single band of amplicon, without primer dimers and at the targeted sizes. Through qRT-PCR, GAPDH gives single peak in dissociation curve in all plant parts used further fulfilling the characteristic of reference genes.Previous work on validation of reference genes in pepper shows that only UBI-3 suits to C. annuum var MC11 infected CMV, thus we suggest that GAPDH has a potential to be a validated reference gene for C. annuum var MC11 and can be used together UBI-3 for the purpose of data normalization. 

Identification of Suitable Reference Genes for Gene Expression Studies in Tissues from Fructose-Fed Rats

2012 ◽  
Vol 5 (2) ◽  
pp. 560-565 ◽  
Author(s):  
Ana Djordjevic ◽  
Danijela Vojnović Milutinović ◽  
Nikola Tanić ◽  
Biljana Bursić ◽  
Ana Vasiljević ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference
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